Templated native silk smectic gels

ABSTRACT

One aspect of the present invention relates to a method of preparing a fibrous protein smectic hydrogel by way of a solvent templating process, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; and collecting the resulting fibrous protein smectic hydrogel and allowing it to dry. Another aspect of the present invention relates to a method of obtaining predominantly one enantiomer from a racemic mixture, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; allowing the enantiomers of racemic mixture to diffuse selectively into the smectic hydrogel in solution; removing the smectic hydrogel from the solution; rinsing predominantly one enantiomer from the surface of the smectic hydrogel; and extracting predominantly one enantiomer from the interior of the smectic hydrogel. The present invention also relates to a smectic hydrogel prepared according to an aforementioned method.

GOVERNMENT SUPPORT

The invention was made with support provided by NASA (grant NAG8-1699)and NSF (grant BES 9727401); therefore, the government has certainrights in the invention.

BACKGROUND OF THE INVENTION

There are broadly three different types of liquid crystalline material:nematic, cholesteric, and smectic. The types are distinguished bydifferences in molecular ordering. Such materials only show a liquidcrystal phase over a limited temperature range between the solid andisotropic liquid phases. Within the liquid crystal phase temperaturerange, a material may exhibit one or more of the nematic, cholesteric orsmectic phase types. Normally, a material is chosen such that it formsonly one type of liquid crystal phase over its working temperaturerange.

Liquid crystalline elastomers combine the various broken symmetries ofliquid crystalline phases with the elasticity of polymer networks. Oneobvious effect is that the single crystal elastomers can undergospontaneous shape changes when they undergo a liquid crystallinetransition. There are many more subtle effects in the interplay betweenthe fluctuations of the familiar liquid crystalline and elastic degreesof freedom (for example, and perfect single crystal nematic elastomercan, in theory, exhibit elastic moduli of zero when deformed in certaindirections. These dramatic effects are, of course, drasticallyinfluenced by the disorder, which makes them perfect for studyingquenched disorder.

Biopolymer networks are found all over nature. For example, thecytoskeleton is supported by a network of actin, which is asemi-flexible polymer with globular proteins as a monomer unit. Thesenetworks are proving to be ideal model systems for understanding thephysics of semi-flexible polymers, both in solution and cross-linkednetwork states.

The fibrous proteins can be considered to be a special class of proteinsthat serve important structural functions in the extracellularenvironment. In living organisms some of these proteins, such ascollagens, are found in thin layers, sandwiched between otherextracellular biomaterials. When studying the physical chemistry ofextracellular fibrous proteins in vitro the use of a two-dimensionalthin film or interfacial environment will help the proteinsself-assemble more efficiently by providing a restricted environment incomparison to three-dimensional bulk systems. It is thus beneficial tostudy the synergistic interaction between the behavior of fibrousproteins in dimensionally restricted environments (such as thin films ortwo-dimensional layers) and the generation of structure and long rangeorder through self-assembly.

Different conformations can be stabilized by an interface, such as anextended chain β-sheet conformation, which maximizes the protein'sspreading and surface area. If the protein or model polypeptide hashydrophobic side chains, and can readily take on a stable α-helicalconformation, α-helices will be stable at the interface. Biridi, K. S.Journal of Colloid and Interface Science 1973, 43, 545; Cheesman, D. F.;Davies, J. T. Advan. Protein Chem. 1954, 9, 439; Jacuemain, D.; Wolf, S.G.; Leveiller, F.; Lahav, M.; Leiserowitz, L.; Deutsch, M.; Kjaer, K.;Als-Nielsen, J. Journal of the American Chemical Society 1990, 112,7724-7736; Loeb, G. I. Journal of Colloid and Interface Science 1968,26, 236; Loeb, G. I. Journal of Colloid and Interface Science 1969, 31,572; Macritchie, F. Adv. Coll. Int. Sci. 1986, 25, 341-382; Magdassi,S.; Garti, N. Surface Activity of proteins; Magdassi, S.; Garti, N.,Ed.; Marcel Dekker: New York, 1991; Vol. 39, pp 289-300; Malcolm, B. R.Nature 1962, 4195, 901; Malcolm, B. R. Soc. Chem. Ind. London 1965, 19,102; Malcolm, B. R. Progress in Surface and Membrane Science 1971, 4,299; Murray, B. S. Coll. Surf A 1997, 125, 73-83; Murray, B. S.; Nelson,P. V. Langmuir 1996, 12, 5973-5976; Weissbuch, I.; Berkovic, G.;Leiserowitz, L.; Lahav, M. Journal of the American Chemical Society1990, 112, 5874-5875; Wustneck, R.; Kragel, J.; Miller, R.; Wilde, P.J.; Clark, D. C. Coll. Surf A 1996, 114, 255-265. The influence of sidechain character in stabilizing an interfacial conformation suggests thathydropathicity can be used as a determinant for interfacialconformation. Carrying this idea further, if a sequence of residuesresults in particular conformations that could exhibit surfactantbehavior, these conformations should be stabilized at an interface.

Silks, and their analogues, have recently been the focus of interest forapplications in biomaterials because of the intriguing properties of thesilk fiber. The simplicity of their sequences lends them to be used asmodel fibrous proteins. Most of the studies on the properties of silks,as well as other fibrous proteins either examine gross materialsproperties such as mechanical properties, thermal stability and surfaceroughness or examine very localized chemical details in the molecule.Literature on long-range ordered “helicoids” is less abundant.

Previously we have disclosed that with B. mori silk fibroin, a threefoldhelical polyglycine II or polyproline II type of conformation wasstabilized by the interface, even though it is not observed in bulk.Valluzzi, R.; Gido, S. P. Biopolymers 1997, 42, 705-717; Valluzzi, R.;Gido, S.; Zhang, W.; Muller, W.; Kaplan, D. Macromolecules 1996, 29,8606-8614; Zhang, W.; Gido, S. P.; Muller, W. S.; Fossey, S. A.; Kaplan,D. L. Electron Microscopy Society of America, Proceedings 1993, 1216.The B. mori fibroin crystallizable sequence is approximately(Gly-Ala-Gly-Ala-Gly-Ser)_(x), and a left-handed threefold helicalconformation, which is sterically reasonable, separates hydrophobicalanine and hydrophilic serine residues to opposite sides of theinterface. Valluzzi, R; Gido, S. P. Biopolymers 1997, 42, 705-717;Valluzzi, R.; Gido, S.; Zhang, W.; Muller, W.; Kaplan, D. Macromolecules1996, 29, 8606-8614; Zhang, W.; Gido, S. P.; Muller, W. S.; Fossey, S.A.; Kaplan, D. L. Electron Microscopy Society of America, Proceedings1993, 1216.

As a consequence of the difficulties entailed in attempting detailedsurface measurements at a liquid-liquid interface, there have been fewstudies on the behavior of proteins at these interfaces to date. Murrayand Nelson, working with a novel oil-water trough design, have publishedresults on the comparative behavior of β-lactoglobulin and bovine serumalbumin (both globular) protein films at air-water and oil-waterinterfaces that appear consistent with structural results obtained forfibrous proteins at air-water and oil-water interfaces. Murray, B. S.Coll. Surf A 1997, 125, 73-83; Murray, B. S.; Nelson, P. V. Langmuir1996, 12, 5973-5976. They found that films at the oil-water interfacewere more expanded and also more expansible and compressible thancorresponding films at the air-water interface. This was believed to bedue to a reduction in aggregation. The increased solubility of thehydrophobic groups in oil as opposed to air is cited as a reason for thegreater stability of films at the oil-water interface. Shchipunov hasstudied phospholipids at an oil water interface, and observed that thepresence of the amphiphiles results in more oil on the water side of theinterface and more water on the oil side. Shchipunov, Y. A.Liquid/Liquid Interfaces and Self-Organized Assemblies of Lecithin;Shchipunov, Y. A., Ed.; CRC Press: Boca Raton, Fla., 1996, pp 295-315.The amphiphile compatibilizes the two liquids forming the interface, andin the process, the interface thickens. Both the compatibilizationeffect observed for the phospholipids and the stability observed for theprotein films suggest that there is oil and water closely interactingwith the side chains of the protein. Side chain—side chain interactionswould thus be expected to be screened. Jacuemain, D.; Wolf, S. G.;Leveiller, F.; Lahav, M.; Leiserowitz, L.; Deutsch, M.; Kjaer, K;Als-Nielsen, J. Journal of the American Chemical Society 1990, 112,7724-7736; Malcolm, B. R. Nature 1962, 4195, 901; Murray, B. S. Coll.Surf. A 1997, 125, 73-83; Murray, B. S.; Nelson, P. V. Langmuir 1996,12, 5973-5976; Wustneck, R.; Kragel, J.; Miller, R.; Wilde, P. J.;Clark, D. C. Coll. Surf. A 1996, 114, 255-265; Shchipunov, Y. A.Liquid/Liquid Interfaces and Self-Organized Assemblies of Lecithin;Shchipunov, Y. A., Ed.; CRC Press: Boca Raton, Fla., 1996, pp 295-315;Miller, I. R. Progress in Surface and Membrane Science 1971, 4, 299.

An aqueous-hexane interface was chosen as an initial probe of fibroinliquid-liquid interface behavior. This interface, in the absence offibroin, is believed to be about 10 Å thick Carpenter, I. L.; Hehre, W.J. Journal of Physical Chemistry 1990, 94, 531-536; Michael, D.;Benjamin, I. Journal of physical Chemistry 1995, 99, 1530-1536. The silkat the aqueous-hexane interface forms a film as it ages, and this filmcan be picked up onto sample grids for observation in a transmissionelectron microscope (TEM). The hexane was expected to be a bettersolvent for the alanine residues in silk than the water, forcing them tothe hexane side of the interface. The aqueous phase should be a bettersolvent for serine.

SUMMARY OF INVENTION

In one embodiment, the present invention relates to a method ofpreparing a fibrous protein smectic hydrogel by way of a solventtemplating process comprising the steps of:

-   -   a. pouring an aqueous fibrous protein solution into a container        comprising a solvent that is not miscible with water;    -   b. sealing the container and allowing it to sit at about room        temperature overnight; and    -   c. collecting the resulting fibrous protein smectic hydrogel and        allowing it to dry.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the solvent is chloroform.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the solvent is iso-amyl alcohol.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the solvent is hexane.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein is selected fromthe group consisting of silk, collagens, keratins, actins, chorions, andseroins.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein is silk.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 4% by weight.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 8% by weight.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 4% by weight, the fibrous protein is silk, and thesolvent is iso-amyl alcohol.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 8% by weight, the fibrous protein is silk, and thesolvent is iso-amyl alcohol.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 4% by weight, the fibrous protein is silk, and thesolvent is chloroform.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 8% by weight, the fibrous protein is silk, and thesolvent is chloroform.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 4% by weight, the fibrous protein is silk, and thesolvent is hexane.

In a further embodiment, the present invention relates to the abovesolvent templating process, wherein the fibrous protein solution isgreater than about 8% by weight, the fibrous protein is silk, and thesolvent is hexane.

In another embodiment the present invention relates to a method ofobtaining predominantly one enantiomer from a racemic mixture,comprising the steps of:

-   -   a. pouring an aqueous fibrous protein solution into a container        comprising a solvent that is not miscible with water;    -   b. sealing the container and allowing it to sit at about room        temperature overnight;    -   c. allowing the racemic mixture to diffuse into the smectic        hydrogel in solution;    -   d. removing the smectic hydrogel from the solution;    -   e. rinsing predominantly one enantiomer from the surface of the        smectic hydrogel; and    -   f. extracting predominantly one enantiomer from the interior of        the smectic hydrogel.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein is selected from the group consisting of silk, collagens,keratins, actins, chorions, and seroins.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein is silk.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein solution is greater than about 4% by weight.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein solution is greater than about 8% by weight.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein solution is greater than about 4% by weight and the fibrousprotein is silk.

In a further embodiment, the present invention relates to the abovemethod of obtaining predominantly one enantiomer, wherein the fibrousprotein solution is greater than about 8% by weight and the fibrousprotein is silk.

In another embodiment, the present invention relates to the fibrousprotein smectic hydrogel prepared by the above solvent templatingmethod.

In a further embodiment, the present invention relates to the fibrousprotein smectic hydrogel prepared by the above solvent templatingmethod, wherein the fibrous protein is selected from the groupconsisting of silk, collagens, keratins, actins, chorions, and seroins.

In a further embodiment, the present invention relates to the fibrousprotein smectic hydrogel prepared by the above solvent templatingmethod, wherein the fibrous protein is silk.

In a further embodiment, the present invention relates to the fibrousprotein smectic hydrogel prepared by the above solvent templatingmethod, wherein the fibrous protein smectic hydrogel is greater than orequal to about 38 nm thick.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the surface (right) and fracture surface of chloroformtemplated silk. The wavy texture is everywhere on the solvent templatedside of the materials surface.

FIG. 2 depicts the chloroform templated film. Waves reorienting andbecoming terraces can be seen, behavior which is not expected for simplewrinkles due to contraction.

FIG. 3 depicts a regular pattern of nubby small structures comprisingthe waves.

FIG. 4 depicts an amyl alcohol film showing a surface that looks like a“nonwoven woven” fabric.

FIG. 5 depicts a surface texture seen at an angle showing a thin layervery different from the chloroform films.

FIG. 6 depicts amyl alcohol templated samples soaked in bipyridyltrisRuII chloride hexahydrate giving a high magnification image and a 40nm layered feature.

FIG. 7 depicts films after soaking in a dysprosium chloride solution foradded contrast. The wavy layered structure of the chloroform templatedfilm is apparent here.

FIG. 8 depicts a film's texture that is even and regular.

FIG. 9 depicts self-fabricated textured “tapes” from a peptide withsequence(Glu)₅(Ser-Gly-Ala-Gly-Val-Gly-Arg-Gly-Asp-Gly-Ser-GlyVal-Gly-Leu-Gly-Ser-Gly-Asn-Gly)₂(Glu)₅. 1.Optical micrograph shows a ˜10-15 micron texture which persists throughthe material thickness. The material is optically transparent. 2.Polarizing optical microscopy reveals patterned birefringence,indicating that the topographic texture is due to a changing materialorientation. 3. SEM image shows the topographic structure of the tape.The difference in periodicity observed in SEM and optical microscopy isdue to the fact that top surface and bottom surface ridges are bothobserved in the optical image (resulting in an apparently shorterperiod).

FIG. 10 depicts self-fabricated tapes of(Glu)₅(Ser-Gly-Ala-Gly-Val-Gly-Arg-Gly-Asp-Gly-Ser-Gly-Val-Gly-Leu-Gly-Ser-Gly-Asn-Gly)₂(Glu)₅have “patterns within patterns” or a long-range ordered structureconsisting of hierarchical nanoscale to microscale patterns; 1: theself-limited width and thickness of the fibers (˜120 microns, 50 micronsrespectively) form the largest length scale in the hierarchy; a 40micron periodic texture is observed running along the tape; 2: withinthe ridges of the 40 micron texture a 3 micron subtexture is observed;3: a submicron texture of inclined sheets or layers can be observed (<40nm, but exact size is below the resolution of the scanning electronmicroscope); TEM studies indicate a layer spacing of ˜5 nm.

FIG. 11 depicts an IR spectra of self-fabricated tapes of(Glu)₅(Ser-Gly-Ala-Gly-Val-Gly-Arg-Gly-Asp-Gly-Ser-GlyVal-Gly-Leu-Gly-Ser-Gly-Asn-Gly)₂(Glu)₅.Typically IR spectra for molecules are seen as very small differences inIR transmission relative to a large backround, which must be subtractedout Raw data (no background subtraction) is shown for transmission FTIRspectra through different regions (orientations) of the tape structure.Two orientations show very typical protein absorbance spectra over ahigh background. However in some orientations the IR radiation does notreach the detector.

FIG. 12 depicts an IR spectrum modified by tape with scale expanded toshow spectral features. Instead of an absorption or transmissionspectrum, a pattern of 2 overlaid sinusoids (one has a 50/cm period, theother a 25/cm period. The effect for this material appears strongest inthe 1750-3500 cm⁻¹, or 5.7-2.9 micron range.

FIG. 13 depicts twisted polycrystals obtained by salt precipitation ofan oligopeptide with Na-EDTA.

FIG. 14 depicts ordered “corkscrew” polycrystalline oligopeptide saltprecipitate as a hierarchy of twisted ordered structures.

FIG. 15 depicts reflection and transmission FTIR spectra for orderedpolycystals. TOP: reflection infrared spectrum, Raw data. A glassydisordered material of the oligopeptide is more reflective than thebackground. An ordered periodic nanolayered material from the samepeptide is shown, and clearly reflects far less of the infraredradiation. BOTTOM: transmission spectra for background, unorderedpeptide material and a chemically identical nanolayered ordered materialof the peptide. Spectrum is greatly attenuated for the ordered material.

FIG. 16 depicts ordered textured surfaces and interiors from templatedgels (a) chloroform templated gels have a wavy surface texture coveringthe surfaces which were in contact with water; (b) a fracture surfacefrom the chloroform templated gel reveals a “skin” of the wavy pattern,which forms channels down into the interior, the interior has adifferent structure, which appears to be made of wavy plates; (c)templated surface of amyl alcohol templated material (in contact withwater); (d) higher magnification image of the edge of the region in c,showing a “skin” core structure and a patterned texture throughout thematerial; (e,f) amyl alcohol dried film after swelling in an aqueoussolution of ruthenium compound and extraction of ruthenium compound byswelling in water; (e) wavy lines indicate reorientation of orderedstructures within the material; (f) at high magnification (20,000×)lines 38 nm in width are observed.

FIG. 17 depicts amyl alcohol templated gel after soaking in AqueousTris(2,2′-bipyridyl) dichloro ruthenium(II) hexahydrate (“Rubipy”)solution for 1 day. Much of the Rubipy has migrated from the solutioninto the silk gel. Initial migration is rapid and chirally selective(occurs over roughly 1 hour). Additional migration occurs slowly afterthis for roughly 1 day and is less chirally selective. Chloroformtemplated gels do not exhibit complex diffusion behavior and arechirally selective throughout the swelling process.

FIG. 18 depicts a cross section of amyl alcohol templated gel afterswelling in Rubipy for 1 hour. The Rubipy penetrated rapidly into theouter “skin” layers of the gel (bright orange), and more slowly into theinterior (yellowish region).

FIG. 19 depicts an X-ray diffraction pattern from chloroform templatedgel. Dark arcs along the diffraction rings (arrow) indicate orientation.

FIG. 20 depicts the non-globular nature of fibrous proteins.

FIG. 21 depicts the long range order of liquid crystals.

FIG. 22 depicts “frustration” in nanolayered crystals.

FIG. 23 depicts nanocomposites.

FIG. 24 depicts banded structures from native silk.

FIG. 25 depicts banded structures from engineered protein designedpeptide.

FIG. 26 depicts how hairpin structures allow silk liquid crystallinity.

FIG. 27 depicts spider silk modification.

FIG. 28 depicts amphiphilic spider silk motif.

FIG. 29 depicts silkworm silk peptide models.

FIG. 30 depicts film morphology and helix anchoring.

FIG. 31 depicts the templating-against-solvent technique.

FIG. 32 depicts patterned peptide films.

FIG. 33 depicts silk templated gels-surface “skin”.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “smectic” is art-recognized and refers to the mesomorphic phaseof a liquid crystal in which molecules are closely aligned in a distinctseries of layers, with the axes of the molecules lying perpendicular tothe plane of the layers.

The term “gel” is art-recognized and refers to a colloid in which thedisperse phase has combined with the dispersion medium to produce asemisolid material.

The term “hydrogel” is art-recognized and refers to a colloid in whichthe particles are in the external or dispersion phase and water is inthe internal or dispersed phase.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring can be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, azetidine,azepine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring can be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo undesired transformation, such as byrearrangement, cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, it maybe isolated using chiral chromatography methods, or by derivation with achiral auxiliary, where the resulting diastereomeric mixture isseparated and the auxiliary group cleaved to provide the pure desiredenantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Fibrous-Protein Smectic Hydrogels

We have demonstrated control over helicoid structure—the materialsuperstructure generated by an array of twisting molecules (like achiral liquid crystal) in a model fibrous protein (silk). The processesused are very simple and can be applied easily elsewhere to createnanostructured “designer” biomaterials for studies in areas ranging fromcell biology and surface interactions to surface nanofluidics. Thesesimple processes were carefully designed using what we know aboutfibrous proteins (as different from synthetic polymers and globularproteins), and their success underscores the possibilities formanipulating these molecules with processes that are tailored forfibrous proteins (rather than using polymer techniques that destroytheir structure, or trying to get them to behave like globularproteins). The characterization results highlight a few key featuresthat distinguish the nature of these proteins.

Remarkably, two processes have been developed that allow the creation ofhighly structured biomaterials from regenerated silks. A silksolubilization process has been modified to obtain concentrations inexcess of 8 wt % silk in aqueous solution. An earlier process yieldspurified silk solutions of up to only about 4-5 wt %, depending on thepurity of the solution and freshness of the raw silk used. A solventtemplating process yields nanostructured permselective materials fromaqueous silk solutions with concentrations of >about 4 wt %. In generalthe solvent templating process yields thicker, bulk solids with the thinfilm features (many layers stacked up) when using about an 8 wt %aqueous silk solution as opposed to about a 4 wt % solution which yieldsa thin film. In tandem, these two discoveries allow the creation ofprotein membranes, films, and gels which are made of discrete stacks ofprotein layers which can be about 38 nm or thicker. Wrinkling andperforation of these layers, combined with chiral stacking interactions(a tendency to twist) result in a number of different very regularmicroscale patterned surface textures. Many of the films selectivelyabsorb small molecules and ions from solution, and many are chirallyselective as well. Thus they may find application as therapeutic agentdelivery materials, components in a chiral separations process, matricesfor chiral enzymes and catalysts, and as chiral templates. Themorphology and microstructure of the films can be controlled by choiceof solvent, starting concentration of protein, and environmental factorssuch as temperature, humidity, addition of ether and/or alcohol to theprotein solution, addition of acid to the protein solution, or additionof divalent ionic salts to the protein solution. Altering theseparameters results in different permeation properties for the proteinmaterials, different molecular orientations observed within the films,and different surface topographies. The length scale of the topographicfeatures and the protein nature of the films also suggests applicationsin tissue engineering and cell biology, where microscale and nanoscalepatterns have been shown to strongly influence cell growth,differentiation, and tumogenesis. The data gathered to date suggest thatthe materials may be chemically patterned as well, allowing bioactivesites and molecules to be precisely placed on the material surface andthroughout the material. This arrangement would result in extremelypredictable and reproducible diffusion rates out of the material fortherapeutic agent delivery applications, as well as suggesting novelsurgical materials patterned to address cellular processes involved inhealing. The material fabrication process is based on chemical andphysical features common to many fibrous protein molecules such ascollagens, keratins, actins, chorions, seroins, and other silks. Many ofthese features are also found in non-protein biopolymers such ascellulose, many polysaccharides, and nucleic acids. We would thus expectthe process to be useful in making patterned biocompatible nanomaterialsfrom a large number of natural molecules in addition to silks.

Silk-Based Smectic Gels

Concentrated solutions (in general about 4 wt % for thin films, about 8wt % for bulk solids comprising several layers stacked up) of silk canbe used to grow hydrogels from an aqueous organic, liquid-liquidinterface. For example, a silk solution is placed into a vessel, asolvent such as chloroform, hexane, or amyl alcohol is carefully layeredon top of the silk solution (underneath in the case of chloroform, whichis denser than aqueous solutions). The layered liquid is covered toprevent evaporation and excessive competing interactions with air, and afilm forms at the interface. In the case of bulk solid hydrogels, thefilm grows into the aqueous silk phase. Solvent templated processing ofnatural silks results in the formation of a nanolayered structure, wherethe layer thickness and chemistry within the layers is determined by thefolding pattern induced in the silk molecules (or other fibrous proteinmolecules) through processing. The nanolayered protein materialstructure is obtained from high concentration solutions of protein,where the molecule and solution may have locally ordered structure priorto templating.

Highly structured templated solid materials cannot be obtained for silksbelow concentrations of about 4 wt % protein, and the most organized andoriented structured materials are obtained from solutions with proteinconcentrations of greater than about 5 wt %. Furthermore, standard filmcasting techniques do not yield ordered solids, even when the startingsolutions contain about 5-8 wt %/o protein. The choice of solvent usedin templating is also important. Solvents which are not at all misciblein water, such as hexane, do not template hydrogels, but instead formviscous liquid crystalline films which are localized in a very thinregion at the interface. Solvents which do not have a greater affinitythan water for some of the side chains in silk (or other protein used)do not result in templating behavior. An example is dichloroethane,which has a low affinity for both the polar and nonpolar side chains ina typical protein and exhibits no surface templating behavior. Solventswhich are somewhat miscible in water, or which make a low energyinterface with water are weakly templating. For example, using propanoland butyl alcohol with a silk solution results in a loose, poorlylocalized gel, due to the large thickness and weak chemical gradient inthe interfacial region. Dried material from these gels is poorlyoriented, not well-ordered, and either does exhibit the pronouncedpermselective properties and microstructure observed in templated films,although weak versions of these properties are sometimes observed.

The choice of salt in the aqueous silk solution is also important. Aswitch from LiSCN to LiBr enabled the preparation of the higherconcentrated silk aqueous solution used for templating ordered bulksolids.

The result of a molecular design which consists of a largeself-fabricating unit and smaller solubilizing/functional ends, is thatthe thermodynamically favorable state for the entire molecule will besimilar to the thermodynamically favorable state for theself-fabricating block. There may be a structural compromise due to thepresence of the end blocks, but since the fabricating block dominatesthe mass and volume of the molecule these compromises are expected to beminor. However, the situation for the solubilizing/functional end blocksis quite different. In a molecular packing geometry dominated byinteraction between self-fabricating blocks the local packing in regionscontaining chain ends will often be highly strained due to thermodynamicfrustration. If the ideal thermodynamically favorable geometry for theend blocks is not compatible with the packing favored by the selffabricating blocks (which comprise most of the molecule) the end blockswill be forced into a state that is far from their (local) thermodynamicideal, and will be “frustrated”. By designing multiblock “miniblock”oligomers with block to block disparities in residue size, volume,preferred conformation, etc. we can design frustrated smecticallyordered solids, where the density and interaction behavior in theinterlayer region is strongly perturbed with respect to bulk material ornon-frustrated surfaces with the same composition.

The use of smectic forming self-fabricating blocks, oligomeric molecularweight and associated liquid to solid transitions, and a nanoscopicdesigned frustrated interlayer region (from end blocks) allows us toconstruct molecularly designed materials with nanoscale fluid channels.These channels are essentially the endblock-rich regions in themultilayered smectic generated structures. Through engineeredmismatching of the properties of monomers used to specify the end blocksvs. the self-fabricating blocks, different properties in these channelscan be designed in. The regions in question contain chain ends and arethus somewhat less constrained than the regions comprised ofself-fabricating blocks. The chain ends protruding into the interlayerregion create a brush at the molecular scale. Molecules absorbed intothe miniblock derived material will migrate preferentially into theinterlayer regions because:

1. Space exists or can be made to accommodate additional molecules(through localized swelling in the interlayer region).

2. Thermodynamic frustration can be alleviated by adding molecules,changing the overall chemistry and preferred state of the region.

3. Strong interactions between self-fabricating blocks precludeincorporation of additional molecules.

4. Interactions and properties designed into the endblocks promotelocalization of an added molecule (solute) into the interlayer region.

Designed interactions can include acid endblocks to attract and localizebasic solutes, low amino acid volumes in the endblocks to attract solutemolecules that balance the interlayer volume and density, matchedendblock-solute hydrophobic/hydrophilic interactions. It is important tonote (and a key feature) that the “solute localizing” propertiesdesigned for the end blocks need not be entirely enthalpic (chemicalinteractions) in nature, but can include entropy-based design ideas aswell (volume, molecule shape, flexibility).

Molecules absorbed into these designed materials (from designedmolecules) will interact with a densely packed “brush” of end blocks,and the strength and nature of this interaction will determine whether asolute molecule can enter the material and diffuse into the materialinterior. If the endblocks are chiral, a chiral interaction occursbetween solute and the nanobrush within the material for every fewAngstroms of diffusion. Even non-specific interactions are expected tobe chirally selective for diffusion of enantiomers through the brush.The extremely large surface area provided by the brush for interactionsprovides high selectivity, the possibility of a largely entropy-drivendesigned diffusion and interaction process ensures that separation isnot specific to a particular well matched solute-endblock pair.

Separation has been observed for a test chiral molecule in silk-like andcollagen-like designed oligopeptides. Acid base interactions were usedto localize the test molecules in the chain end regions. Two processeswere used to absorb the test molecule into the material:co-self-assembly from solution and swelling of an assembled miniblockoligopeptide nanomaterial with a solution of the test molecule. Bothprocesses result in chiral separation, but smectic or higher level orderis required in the oligopeptide nanomaterial to achieve good results.Thus we can elucidate some key design features for chiral separationusing these materials:

1. Robust smectic layer formation.

2. Functional blocks used to localize solute in interlayer region(enthalpiclly or entropically).

3. Chiral functional blocks forming nanoscale chiral pores or interlayerbrushes to provide a high surface area of interaction.

4. Sufficient structure and density in the nanomaterial to preventnon-specific diffusion (smectic or higher order, density comparable tohomopolymer or greater).

5. Chemical compatibility with solute and solvent for solute. Ideallythe nanostructured material should swell in the solvent to promotesolvent diffusion, but not dissolve. Swelling should be limited to <50%increase in the volume of the endblocks (e.g. if endblocks are 20% ofthe material a swelling of not more than 10%).

Chiral enantiomers can be separated by diffusing the racemate into thematerial in solution and then removing the material, rinsing it toremove “bad” enantiomer on the material surface, and solvent extractingthe “good” enantiomer. Alternatively, the material could be used to“sponge” up the undesirable enantiomer leaving the desired enantiomerbehind. As yet another possible separation process, the material couldbe made into a membrane which would allow only one enantiomer to passthrough.

Materials can also be designed (at the molecular level) for lessdemanding applications than chiral separations. Simple achiral chemicalselectivity can be incorporated for permselective membranes andseparation beads. The designability of both molecules and materials alsoallows selection based on size, through design of the size of layer andsublayer features (>2 nm particles filtered), and through design oflayer densities using mismatched monomer sizes in the oligomer blocks tocreate molecular scale porosity.

Other applications for a chirally separating material extend beyondchiral separations to include chiral catalysis, enzyme substrates, andother combined chemical separation and reaction processes. For example achiral enzyme might experience enhanced chiral selectivity in a chiralenvironment due to chirally differentiated constraints on the diffusionand reorientation modes of reactants. Different activated states ofreactants and different conformational states of a chiral catalyst wouldbe expected to be preferentially stabilized in an environment withchiral physical features on the length scale of a molecule when comparedto a more symmetric environment. At the surface of a chiralnanopatterned material, a loosely bound enzyme (for example tethered toa swollen hydrophilic nanolayer by a single covalent bond) wouldexperience an environment which has features of homogeneous catalysis.The enzyme would be surrounded by an essentially fluid “gel” where asharp symmetry breaking solid surface is not defined. sorption to asolid surface) nor constrains the geometry of reactant approach.However, the enzyme would nevertheless be bound to the material andrecoverable. Furthermore the fluid (of solubilizing ends or blocks)surrounding the enzyme would be densely packed and chiral, encouragingchiral interactions to stabilize different enzyme conformations (whencompared to a symmetric environment).

Along similar lines a chiral catalyst for a polymerization could beembedded in the chiral nanomaterial membrane. Chirally biased transportof monomers and stabilization of a preferred chirality (for monomersthat readily racemize) could be used to direct catalysis and subsequentregularity/purity or the product polymer or other reaction product.

Samples

A set of samples have been prepared from concentrated solutions ofnatural silk using a solvent templating technique. These samplesinitially formed as hydrogels which grew from a solvent interface intothe aqueous silk phase. These hydrogels lose more than 90% of theirvolume on drying. Comparisons of dried gels prepared using chloroform asthe templating solvent to dried gels prepared using amyl alcohol havebeen made. In X-ray studies (WAXS), the gels prepared using chloroformare oriented biaxially whereas the amyl alcohol gels have a weakuniaxial orientation. Orders of a 100-110 Angstrom layer spacing asfaint blips on top of the WAXS pattern are also observed. The layerspacing in synchrotron SAXS has not been reproduced, but at this time itis not known whether the low angle spacing is imaginary (an artifact ofthe detector design) or whether its absence is due to a combination oflow exposure times in the synchrotron beam, sample orientation and largeoriented domain sizes, and the one dimensional detector at the beamline. Small features (very regular) of the same size are observed inFESEM.

FTIR data on either film was not able to be obtained at this time. Intransmission and reflection IR experiments, most of the raw signalrequired to obtain a spectrum (a difference spectrum between raw signaland background) is lost This spectral dropout occurs in the wavelengthregion from 2 and 8 microns. In ATR one does not see any spectrum or anysignificant raw signal using ZnSe as the ATR crystal. Since ATR shouldallow one to see any material that is really there, and ATR fromsimilarly problematic films have been obtained when a Si cell was used,we speculate that the films strongly polarize infrared radiation in thiswavelength region. The ZnSe crystal used is a hexagonal, randomlyoriented material and is thus optically anisotropic—it induces apolarization state in the incident infrared radiation. Silicon is cubicand is optically isotropic. In addition to polarizing the infraredradiation, the samples have no spectrum in diffuse reflectance IR (thusruling out Bragg diffraction), indicating that they behave as completelyIR absorptive little black bodies even when ground into a fine powder.The grain size of the powder (a few microns) can be used to place anupper limit on the smallest film thickness required to obtain a proteinblack body in the infrared. This may correspond to one full cycle ofsome chiral feature in the films, but it is also possible that a partialfeature has the same effect. This is similar to a related phenomenonwhere dense clusters in an inhomogeneous material preferentially absorbinfrared radiation, resulting in a lower total signal for the materialin the clusters, and spectral information weighted towards the lessdense material. This phenomenon is relevant to the difference spectra;we are seeing anomalies in the raw spectra.

Since the structures are all reasonable in terms of chiral liquidcrystalline and polymer phase behavior and microstructure, the observedphenomena are not specific to proteins but can be generalized to anymolecule type, provided that it can be designed to form the appropriateshapes. So as discotic liquid crystalline phases and order are common toall disc shapes molecules, the structures may be general to all polymerswhich can form chiral hairpins and folded structures. Different moleculetypes may give better materials properties and also allow one to pump upthe strength of the relevant chiral interactions and produce eventwistier materials (with analogous morphologies).

The native silk films are comprised of wavy, probably interconnected,layers. FIGS. 1-8 depict SEM images of differently oriented fracturesurfaces, where the edges of the wavy layers can be seen. In otherorientations the morphology looks like a honeycomb of ˜75 nm features.Some ˜11 nm pores in a very regular honeycomb inside the 75 nm featurescan be made out. These may be responsible for the bizarre infraredbehavior observed. The materials show marked differences in surfacemorphology (as well as orientation) depending on templating solvent.There may also be small differences in the phantom layer spacing. Theless ordered amyl alcohol film is a very good ion scavenger. Thechloroform templated film scavenges chiral ions. Both films will soak upenough rare earth salt to become refractive and shiny.

All of the native silk materials will swell very slightly and soften inwater or weak acid, and will scavenge bases and become hard. They areinsoluble in alcohol. They are thermally stable to 290° C. at whichpoint they degrade rapidly without melting. They appear to be fairlytough and hard, and the starting materials and processes are relativelyinexpensive.

Fibrous-Protein Peptide-Based Smectic Gels

Biologically inspired nanopatterned materials have been designed,synthesized (as complex molecules) and fabricated. These materials havesome unusual spectral features in the mid-far infrared. They can befabricated efficiently, combined with inorganics and salts to createnanocomposites (to modify specific properties), fabricated frommolecules which can be synthesized in quantity, and have reasonablemechanical and thermal properties. The materials incorporate arepetitive nanoscale pattern of chemistry and molecular orientationwhich persists to macroscopic length scales (in some cases millimetersor centimeters in initial studies). The current focus is on nanoscalematerial patterns which incorporate a small nanoscale multilayeredstructure which is achieved through molecular design and self-assembly,but other types of geometric nanoscale patterns are observed andfabricated as well. Engineering of all of the geometric features (andmany chemical features) of the nanoscale pattern is possible throughmolecular level design.

The materials are peptide-based, and several distinct classes ofpolypeptides and oligopeptides have been defined which are loosely basedon natural fibrous proteins such as collagens, keratins, and silks. Theindividual oligopeptides within each class incorporate simplifiedversions of patterned amino acid motifs found in each protein type(collagen, keratin, silk) with designed variations included to enablestudy of very specific molecular level influences on folding, materialself-assembly, and resulting materials properties. A strong interest inthe design of these oligopeptides has been the creation of modelmolecules that allow us to utilize liquid crystalline behavior indesigning simple robust approaches to chemical and physical patterningof materials at the micro- and nano-scales. A key feature of thisapproach as mentioned previously includes designing molecular materialswhich segregate to form nanoscale long-range ordered patterns as athermodynamically favorable state, often through built-in molecularchemical complexity resulting in thermodynamic “frustration”. A majoravenue being studied to form materials from these molecules ismanipulation of a folding or aggregation transition which allows themolecule to change it's liquid crystalline self-assembly behavior fromflexible soap-like lyotropic liquid crystallinity to chiral orientedrigid rod thermotropic liquid crystalline behavior. Our ability tomanipulate the transition from one type of behavior to the other givesus significant control over macroscopic features of the material such asdomain size, precipitate shape, etc.

While the materials obtained are biopolymeric in origin, they are nottraditional “folded proteins”, but act much more like synthetic nylons(proteins are very fancy nylons in their chemical backbone structure).They do not form compact natured globules. Instead interactions betweenmolecules are favored, resulting in a molecular solid with reasonablethermal stability, toughness, and strength. Qualitative tests ofmechanical properties indicate that they behave like “good plastics”,having properties similar to nylons. Preliminary thermal analysis in anX-ray beam line suggest that structure is retained to approximately 200°C. for the materials tested thus far. In one of the classes of materialsoptical clarity and optical orientation (a birefringent pattern) wereobserved to persist to 170° C. Variants have been designed withdifferent solubility behavior, and thus considerable control overchemical processability and chemical resistance has been achieved aswell.

Unlike many structural biopolymers and experimental high performancepolymers such as block copolymers, the amino acid sequence and sizerange of our oligopeptides allows facile biosynthesis and scale up.Initial attempts at Biosynthesis have already resulted in high yieldsand scale up routes for the most interesting sequences are beingactively pursued. The intermediate size of the molecules—too small to be“polymers” but too large to be “small molecules”—provides advantages inboth synthesis and processing. The molecules are small by proteinstandards, and thus biosynthesis and scale-up do not present aninsurmountable technical challenge (compare this to the long history ofattempts to biosynthesize high molecular weight collagens and silks).Solubilization of the molecules during purification and processing isalso simplified by their relatively low (for a protein or polymer)molecular weight. They are large molecules when considered asthermotropic liquid crystals, and their size helps to stabilize liquidcrystalline textures while solvent is removed, resulting in liquidcrystalline ordered solids. The chemical complexity of the molecules canbe designed and exploited, allowing each individual sequence to adoptdifferent chemically distinct “states” (which are induced). This abilityto induce a molecule to change it's shape and chemical properties allowsone to engineer irreversible solubility changes into the molecules,making stable materials from processable (under mild conditions)molecules.

Many of the molecules under study exhibit a number of chiral smectic(nanolayered) liquid crystalline phases, which can be dried undercontrolled conditions to create nanolayered materials. An example is thetextured oriented “tape” shown in FIG. 9 (silk-like). These materialsalso posses a hierarchy of patterned features at differentlength-scales, which may be responsible for some of their opticalbehavior. This hierarchical order or patterning is shown in FIG. 10 forthe textured tape. A number of these nanolayered materials have beenstudied, and a common feature for both collagen-like and silk-likematerials is the loss of part of the raw mid-IR spectrum when FTIRspectra are obtained from the materials in a spectrometer. The effect isorientation dependent for many of the materials. In FIG. 11 a set oftransmission spectra are shown. The raw background has the highestintensity. Very thin tape regions in an orientation that does not havean abnormal affect on IR spectra produce very ordinary protein orpeptide IR spectra. In certain orientations we see spectra such as theone represented in FIG. 11.

The regions of the infrared which are strongly affected (thewavelengths) have been correlated to the periodicity of materialsmorphology patterns for one of the classes of oligopeptide materials(collagen-like). Different processing conditions used to make the(silk-like) tapes, used as an example here, also result in differentperiodicities in their morphological texture and in differences in theinfrared wavelength regions affected. There is not enough data on allsamples producing the effect to develop clear correlations betweenprocessing, morphological texture, and infrared behavior for all of thematerials which possess unusual infrared behavior. However, it isbelieved that these correlations exist. Because very normal proteinabsorption spectra can also be obtained from very specific orientationsof the nanolayered materials, or through grinding of the material toreduce the presence and persistence of long range order, the infraredabsorption behavior of these substances is unremarkable at the molecularlevel. However the presence of a long-range ordered pattern of molecularorientations in the self-assembled materials is causing the infraredradiation to miss the spectrometer detector and perhaps go somewhereelse. Because all of the observable materials textures are due tochanges in the local molecular orientation or the orientation ofnanoscale layers in the structure, periodic physical features shouldalso correspond to periodic modulations in the refractive index of thematerial. The raw spectra in the strongly affected regions have a strongsinusoidal character, as can be seen in the rescaled data in FIG. 12(unfortunately, due to resealing the Y-axis intensities are no longerphysically meaningful). In many instances these sinusoidal patterns areattenuated or “chirped” in the small wavenumber/long wavelength part ofthe spectrum. If these materials can redirect or guide radiation in themid—far infrared wavelength region, they may be quite useful inredirecting infrared signatures either to a heat sink, or a detector, ora spectrometer for in-line chemical analysis.

This effect is also observed in layered polycrystals of the sameoligopeptides (collagen-like and silk-like classes) cocrystallized withan organic salt (FIG. 13). These layered polycrystals have individuallathe shaped crystallites arranged in regularly twisting structures(FIG. 14). In this case the infrared effect appears most pronounced forthe most highly organized and regular twisted polycrystalline materials.Attenuation is observed in both reflection infrared spectroscopy andtransmission infrared spectroscopy (FIG. 15). The ordered material isthus both less transmissive and less reflective than the ZnS backgroundin the affected region of the infrared. SAXS data indicate a strongnanoscale layered periodicity (preliminary and unprocessed, not shown).Thermal studies using WAXS and SAXS indicate a slightly lower thermalstability for these salted polycrystalline materials, around 160° C.,when compared to pure oligopeptide materials. Very local phase changesoccur at fairly low temperatures (<100° C.). These local changes can beattributed to extension of the molecules in the nanoscale layers,resulting in a change in layer spacing. Such local phase changes mayprovide useful avenues for manipulating the properties of the materials,but have not been studied in detail yet. The polycrystals arequalitaitively “hard” (as compared to other polymeric crystals) anddifficult to grind (tough). The relatively low thermal stability ofthese polycrystals may be due to the choice of salt—in this case a lowmelting organic molecule. Other salts have produced nanocomposites whichretain the nanolayered structure imposed by the oligopeptides, but whichappear to be more thermally stable than pure peptide, although this datais very preliminary.

The oligopeptide materials can be processed into tough precipitatedtapes, polycrystalline aggregates, or thin films (poorer mechanicalproperties, but we're addressing this). All of these materialsmanipulate the infrared spectra although the silk-like class ofmaterials appear to affect a broader band of the spectrum than thecollagen-like materials. The collagen-like materials affect the 3-10micron region of the infrared most strongly, and have materialperiodicities in the same size range which appear correlated to thespectral band affected. These materials features can be manipulatedthrough selection of sequence patterns in the chemical structure of themolecules, through tuning of the anchoring behavior of the molecules inthe liquid crystalline state during processing, and are expected to alsorespond to a low voltage electrical field applied during formation ofthe liquid crystal and drying to form a solid material. Control ofmaterial texture variations and their correlation to infrared spectralbehavior has not yet been systematically addressed for the silk-likeclass of biopolymer materials (tapes and polycrystals in the examples).However differences are observed depending on the solvent conditionsused to create the tapes, and differences in the region of the infraredaffected are also observed.

EXEMPLIFICATION Example 1 Preparation of Silk

Materials—Cocoons of B. mori silkworm silk were kindly supplied by MTsukada, Institute of Sericulture, Tsukuba, Japan. Chloroform, hexaneand iso-amyl alcohol were purchased from Aldrich and Fisher Scientificand used without further purification.Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (“Rubipy”) waspurchased from Aldrich. Preparation of Regenerated B. mori Silk FibroinSolutions—The silk fibroin solutions were prepared by either one of twomethods: Method A—for preparation of bulk solids—B. mori silk fibroinsolutions were prepared as follows. Cocoons were boiled for 30 min in anaqueous solution of 0.02 M Na₂CO₃, then rinsed thoroughly with water toextract the glue-like sericin proteins. The extracted silk was thendissolved in 9.3 M LiBr solution at room temperature yielding a 20 wt%/o solution. This solution was dialyzed in water using Slide-a-Lyzerdialysis cassettes (Pierce, MWCO 2000) for 48 hrs. The finalconcentration of aqueous silk solution was 8.0 wt %, which wasdetermined by weighing the remaining solid after drying. Milliporepurified water, 17 MΩ, was used throughout all processing. No buffers,acids, or salts were added in final solution; Method B—for preparationof thin films—Bombyx mori silk cocoons were degummed using repeatedwashings in boiling water, sodium dodecyl sulfate (SDS) and NaCO₃ toremove the sericin, leaving pure fibroin. For the first washing 6.5% SDSand 1.0% NaCO₃ were used in boiling water. The cocoons were then rinsedwith 0.4% NaCO3 in boiling water, and subsequently rinsed with boilingwater alone. Other cocoons were degummed without SDS, using only NaCO₃and boiling water. Amino acid analysis has been used to assess theprotein composition of fibroin prepared in this manner and no sericinwas detected. Vallnuzzi, R.; Gido, S. P. Biopolymers 1997, 42, 705-717;Valluzzi, R.; Gido, S.; Zhang, W.; Muller, W.; Kaplan, D. Macromolecules1996, 29, 8606-8614. The degummed fibroin was rinsed thoroughly withdistilled water and dissolved in a 9.1 M solution of LiSCN in water. Inorder to remove the salt, the fibroin and LiSCN solution was thendialyzed against frequent changes of distilled water for several days.The dialyzed fibroin solutions were filtered using a 100 μm syringefilter to remove dust and any protein precipitate.

Example 2 Preparation of Smectic Gels

Preparation of interfacial gels—Aqueous-chloroform, -hexane and-iso-amyl alcohol interfaces were prepared by adding silk peptidesolution into glass vials containing each solvent. The vials were thencapped to prevent evaporation and left at room temperature overnight.The resulting interfacial gels were collected and dried at roomtemperature overnight.

Desalted, HPLC purified, and lyophilized collagen-like peptide wasobtained from the Protein Chemistry Core Facility at the Tufts MedicalSchool. The sequence was (Glu)₅(Gly-Val-Pro-Gly-Pro-Pro)₆(Glu)₅. Theglutamic acid blocks were added to the ends of the peptides to promotesolubility in water so that contaminant salts would not complicateanalysis. Similar peptide design strategies have been used by Rotwarfet. al. to examine the solution behavior of β-sheet forming peptides.Rotwarf, D. M.; Davenport, V. G.; Shi, P.-T.; Peng, J.-L.; Sheraga, H.A. Biopolymers 1996, 39, 531-536. The collagen-like peptide wasdissolved in 18 MΩ Millipore filtered water at a concentration of 1mg/ml peptide in water. No salt or acid or extra reagent was required toaid dissolution. The solution was allowed to stand in an air-tightcapped vial overnight, and then a gold mesh TEM grid (no substrate film)was dipped through the air-water interface.

Example 3 Characterization

Characterization—Gels treated with ruthenium compound were cut to obtaincross sections. The amyl alcohol gel in Rubipy is shown in FIG. 16. Theruthenium compound (Rubipy) has an orange color, and is in the silk gelin a higher concentration (bright red orange) than in the surroundingsolution (light yellow orange). Gels treated with Rubipy for 1 hour weresliced open to obtain cross sections (FIG. 17). These cross sectionsallow us to compare the structure of the gel with a lot of Rubipyabsorbed to the structure of the interior, which has a lowerconcentration of Rubipy. A cross section is shown in FIG. 18, and a darkred-orange Rubipy-rich skin can be seen surrounding a clear yellowishsilk core. Understanding and engineering the skin core morphology formedthrough the templating process is important for controlling thecharacteristics and function of the gels.

SEM—Images of dried gels were obtained with a LEO Gemini 982 FieldEmission Gun SEM. Working distance was 7 mm and applied voltage was 1 to2 kV. All images of gels were taken without any conductive coating.(FIG. 18).

XRD—WAXD experiments were done using Bruker D8 Discover X-raydiffractometer with GADDS multiwire area detector. 40 kV and 20 mA and0.5 mm collimator was used. The distance between the detector and thesample for WAXD was 60 mm. CuKα. Layered structures (6-12 nm layers)with Silk I secondary structures (a non integer helix between the silkII β-strand and the silk m three fold helix) were observed. Thechloroform gels had high orientation in WAXS (FIG. 19).

TEM—Interfacial films of silk fibroin and the peptide were characterizedusing a JEOL 2000 FX-II TEM operating at 200 kV accelerating voltage.Samples were maintained at below −150° C. during the TEMcharacterization, utilizing a cryogenic sample holder. Working atcryogenic temperatures was necessary in order to reduce beam damage andto prevent the loss of water from hydrated crystal structures in thehigh vacuum of the microscope. Electron diffraction and TEM bright fieldimaging were used to assess the structures in the films. An internalgold standard was used to determine lattice spacings. Defocuseddiffraction imaging was used to determine the relative orientations ofthe diffraction patterns and banding or crystallite facets in themorphology images. Detection of salt contamination and thecharacteristics of residual salt crystallites have been described inprevious papers. Valluzzi, R.; Gido, S. P. Biopolymers 1997, 42,705-717; Valluzzi, R.; Gido, S.; Zhang, W.; Muller, W.; Kaplan, D.Macromolecules 1996, 29, 8606-8614. No salt artifacts were observed inthe structures obtained from the water-hexane interface.

Incorporation by Reference

All of the patents, patent applications, and publications cited hereinare hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of preparing a fibrous protein smectic hydrogel, comprising:a. contacting an aqueous fibrous protein solution with a solvent that isnot miscible with water; b. allowing the solution in contact with thesolvent to age at about room temperature or under conditions preventingevaporation or both; and c. collecting the resulting fibrous proteinsmectic hydrogel; and optionally allowing the hydrogel to dry.
 2. Themethod of claim 1, wherein the solvent is chloroform.
 3. The method ofclaim 1, wherein the solvent is iso-amyl alcohol.
 4. The method of claim1, wherein the solvent is hexane.
 5. The method of claim 1, wherein thefibrous protein is selected from the group consisting of silk,collagens, keratins, actins, chorions, and seroins.
 6. The method ofclaim 1, wherein the fibrous protein is silk.
 7. The method of claim 1,wherein the fibrous protein solution is present in greater than about 4%by weight.
 8. The method of claim 1, wherein the fibrous proteinsolution is present in greater than or equal to about 8% by weight. 9.The method of claim 1, wherein the fibrous protein solution is presentin greater than about 4% by weight, the fibrous protein is silk, and thesolvent is iso-amyl alcohol.
 10. The method of claim 1, wherein thefibrous protein solution is present in greater than or equal to about 8%by weight, the fibrous protein is silk, and the solvent is iso-amylalcohol.
 11. The method of claim 1, wherein the fibrous protein solutionis present in greater than about 4% by weight, the fibrous protein issilk, and the solvent is chloroform.
 12. The method of claim 1, whereinthe fibrous protein solution is present in greater than or equal toabout 8% by weight, the fibrous protein is silk, and the solvent ischloroform.
 13. The method of claim 1, wherein the fibrous proteinsolution is present in greater than about 4% by weight, the fibrousprotein is silk, and the solvent is hexane.
 14. The method of claim 1,wherein the fibrous protein solution is present in greater than or equalto about 8% by weight, the fibrous protein is silk, and the solvent ishexane.
 15. The method of claim 1, wherein the smectic hydrogel is abulk solid hydrogel comprising several ordered layers of the fibrousprotein.
 16. A method of obtaining predominantly one enantiomer from amixture of enantiomers, comprising the steps of: a. contacting anaqueous fibrous protein solution with a solvent that is not misciblewith water; b. allowing the solution in contact with the solvent to ageat about room temperature or under conditions preventing evaporation orboth; c. allowing the enantiomers of the mixture to diffuse selectivelyinto the resulting fibrous protein smectic hydrogel in solution; d.removing the smectic hydrogel from the solution; e. rinsingpredominantly a first enantiomer from the surface of the smectichydrogel; and f. extracting predominantly a second enantiomer from theinterior of the smectic hydrogel.
 17. The method of claim 16, whereinthe fibrous protein is selected from the group consisting of silk,collagens, keratins, actins, chorions, and seroins.
 18. The method ofclaim 16, wherein the fibrous protein is silk.
 19. The method of claim16, wherein the fibrous protein solution is present in greater thanabout 4% by weight.
 20. The method of claim 16, wherein the fibrousprotein solution is present in greater than or equal to about 8% byweight.
 21. The method of claim 16, wherein the fibrous protein solutionis present in greater than about 4% by weight and the fibrous protein issilk.
 22. The method of claim 16, wherein the fibrous protein solutionis present in greater than or equal to about 8% by weight and thefibrous protein is silk.
 23. The method of claim 16, wherein the smectichydrogel is a bulk solid hydrogel comprising several ordered layers ofthe fibrous protein.
 24. A fibrous protein smectic hydrogel preparedaccording to the method of claim
 1. 25. The fibrous protein smectichydrogel of claim 24, wherein the fibrous protein is selected from thegroup consisting of silk, collagens, keratins, actins, chorions, andseroins.
 26. The fibrous protein smectic hydrogel of claim 24, whereinthe fibrous protein is silk.
 27. The fibrous protein smectic hydrogel ofclaim 24, wherein the fibrous protein smectic hydrogel is greater thanor equal to about 38 nm thick.
 28. The fibrous protein smectic hydrogelof claim 25, wherein the fibrous protein smectic hydrogel is greaterthan or equal to about 38 nm thick.
 29. The fibrous protein smectichydrogel of claim 26, wherein the fibrous protein smectic hydrogel isgreater than or equal to about 38 nm thick.
 30. The fibrous proteinsmectic hydrogel of claim 24, wherein the fibrous protein smectichydrogel is a bulk solid comprising several ordered layers of thefibrous protein.
 31. A chiral composition comprising a liquidcrystalline ordered solid having a nanoscale multilayered structure,wherein each layer comprises a molecularly oriented fibrous protein, andwherein the layers define an interlayer region having nanoscale chiralpores or channels.
 32. The composition of claim 31, wherein the solid isa hydrogel.
 33. The composition of claim 31, wherein the liquidcrystalline ordering comprises a smectic phase.
 34. The composition ofclaim 31, wherein the liquid crystalline ordering comprises a chiralsmectic phase.
 35. The composition of claim 31, wherein the liquidcrystalline ordering comprises a chiral liquid crystalline phase. 36.The composition of claim 31, wherein the fibrous protein is selectedfrom the group consisting of silk, collagens, keratins, actins,chorions, and serions.
 37. The composition of claim 36, wherein thefibrous protein is silk.
 38. The composition of claim 31, wherein theliquid crystalline order persists to macroscopic length scales on theorder of millimeters or centimeters.
 39. The composition of claim 31,wherein the fibrous protein includes endblocks that promote localizationof a solute molecule added to the composition to the interlayer region.40. The composition of claim 31, further comprising an enzymeincorporated into the chiral composition.
 41. The composition of claim31, further comprising a catalyst incorporated into the chiralcomposition.
 42. A method of obtaining predominantly one enantiomer froma mixture of enantiomers of a chiral molecule, the method comprising: a)contacting the mixture of enantiomers with a chiral compositioncomprising a liquid crystalline ordered solid having a nanoscalemultilayered structure, wherein each layer comprises a molecularlyoriented fibrous protein, and wherein the layers define an interlayerregion having nanoscale chiral pores or channels; and b) isolatingpredominantly one enantiomer within the chiral composition.
 43. Themethod of claim 42, further comprising extracting the enantiomerisolated within the chiral composition.
 44. The method of claim 42,wherein contacting the mixture of enantiomers with the chiralcomposition comprises allowing the enantiomers to diffuse selectivelyinto the chiral composition in solution.
 45. The method of claim 44,further comprising removing the chiral composition from the solution andrinsing predominantly another enantiomer from the surface of the chiralcomposition.
 46. The method of claim 42, wherein the mixture ofenantiomers is contacted with a membrane including the chiralcomposition, and wherein predominantly one enantiomer is isolated withinthe membrane and predominantly another enantiomer is allowed to passthrough the membrane.
 47. An isolated silk protein oriented to providechiral surfaces capable of use as a chiral selector in a chiralseparation.
 48. The use of an isolated silk protein as a chiral selectorin a chiral separation.